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Linking the conformational landscape to enzymatic function through functional site distant mutations

通过功能位点远距离突变将构象景观与酶功能联系起来

基本信息

批准号：
10543155
负责人：
Thomas Michael Sabo
金额：
$ 32.44万
依托单位：
UNIVERSITY OF LOUISVILLE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-01-01 至 2026-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10543155
关键词：
2019-nCoV Active Sites Affect Antineoplastic Agents Architecture Binding Calorimetry Catalysis Cyclic GMP DNA Data Development Dissociation Distant Enzymes Equilibrium Future Generations Grant Guanosine Triphosphate Guanylate kinase Human Kinetics Laboratories Ligand Binding Ligands Link Malignant Neoplasms Measurable Measures Methods Molecular Conformation Motion Muramidase Mutation NMR Spectroscopy Nuclear Magnetic Resonance Nucleosides Nucleotides Outcome Pharmaceutical Preparations Phosphorylation Phosphotransferases Physiological Play Probability Production Proteins Protocols documentation RNA Rationalization Regulation Relaxation Research Personnel Residual state Role SARS coronavirus SUMO1 gene Sampling Scheme Series Shapes Single Nucleotide Polymorphism Site Structural Biologist Structure Testing Therapeutic Thermodynamics Titrations Ubiquitin Variant Vertebral column Viral Work cancer cell conformational conversion design drug discovery enthalpy inhibitor molecular dynamics mutant small molecule inhibitor therapeutic target tool

项目摘要

An ongoing debate concerns the role conformational motions, often termed dynamics, play in biomolceular funtion. For enzymes, it so happens that the timescales for large-scale domain motions are similar to the apparent "$$). catalytic rate (𝑘!"# This observation is where the major point of contention has developed: do "$$. In this context, the proposed studies conformational motions directly impact the true rate of catalysis (𝑘!"#) or 𝑘!"# will further explore how the modulation of the conformational landscape can indeed fine-tune "$$ without 𝑘!"# impacting 𝑘!"# and the ground state structure. The genesis of this proposal arises from our work with human guanylate kinase (hGMPK), a potential therapeutic target for treating cancer and perhaps even SARS-CoV-2, which motivated us to solve the first structure of hGMPK with nuclear magnetic resonance (NMR) spectroscopy (PDB: 6NUI). While solving the hGMPK structure, we expressed a series of seven functional site distant (FSD), "$$ when compared to the wild- non-synonymous single nucleotide variants (nsSNVs) of hGMPK that enhance 𝑘!"# type (wt). Intriguingly, the 2D [1H,15N]-HSQC NMR spectra of the wt hGMPK and its nsSNVs suggest that the 𝑎𝑝𝑝) for GMP binding to FSD mutations minimally impact hGMPK’s backbone fold, yet the apparent off-rates (𝑘𝑜𝑓𝑓 wt and the FSD mutant V91M differ by ~3000 s-1. We hypothesize that hGMPK’s activity can be modulated with FSD mutants by reshaping the conformational landscape. Utilizing NMR spectroscopy and isothermal calorimetry, we will test this hypothesis in the following two Specific Aims. In Aim 1, we will quantify the impact of the FSD mutations on the conformational landscape from kinetic and thermodynamic perspectives. The results from this Aim will provide a comprehensive picture as to where within the hGMPK catalytic and binding schemes the FSD mutations have the largest impact on function. For Aim 2, we will deconvolute the contribution transient structures within the conformational landscape play in enzymatic catalysis through experimentally driven ensemble generation. Our protocol will select hGMPK structures from unbiased molecular dynamics (MD) simulations based on residual dipolar couplings and cross-correlated relaxation rates measured with NMR. The ensembles will aid in the identification of the functionally important transient conformations and an assessment of the impact the FSD mutations have on backbone dihedral correlated motions. To our knowledge, this proposal provides the first examples 1.) of experimentally driven, ensemble generation for an enzyme spanning physiologically relevant timescales and 2.) of determining thermodynamic and kinetic parameters with ligand binding to the same exact site on a series of enzyme variants. The impact of this proposal is the direct linkage of the conformational landscape to enzymatic function. Immediate applications for these results include drug discovery, where targeting structures within the conformational landscape rather than the ground state structure will lead to better outcomes, and biomolecular design, where FSD mutations can be implemented to adjust function through manipulation of the conformational landscape.

构象运动（通常称为动力学）在生物分子动力学中所起的作用一直是争论的焦点